Cd3 alexa fluor 700

CD3-Alexa Fluor 700, CD4-BV605, CD8-APCy7, CD38-APC, CD27-PE-Cy7, CD19-PE-TxR, PD1-PE, ICOS-PcPCy5.5 and CXCR5-AF488 (BD Biosciences).

We used the following conjugated antibodies: CD38-fluorescein isothiocyanate, CD34-phycoerythrin (PE)-cyanin5 (Cy5), CD4-Pacific Blue, CD19-allophycocyanin (APC), CD3-Alexa Fluor 700, CD8-APC-H7, CCR7-Alexa Fluor 647, CD79a-APC from BD Biosciences (San Jose, CA); CD56-PE, CD45RA-PE-TexasRed, CD7-PE-Cy7, CD1a-PE, CD5-PE-Cy5 from Beckman Coulter (Brea, CA); CD31-PE from Miltenyi Biotech (Bergisch Gladbach, Germany) and CD27-PE-Cy7 from eBioscience (San Diego, CA). We obtained LIVE/DEAD aqua fluorescent dye from Molecular Probes (Invitrogen, Carlsbad, CA). The Fam-FLICA-FITC probe against active caspase-1 was used according to the manufacturers’ instructions (ImmunoChemistry, Bloomington, MN). Standard protocols were used for all types of staining. Data were acquired with an LSRII Flow Cytometer (BB Biosciences), and analyzed with FlowJo v7.6.5 (Treestar, Ashland, OR).

For the eight elderly donors, PBMCs were isolated from fresh blood samples using density gradient centrifugation with Ficoll (GE Healthcare). CD3⁺ T cells were enriched from PBMCs by immunomagnetic selection using CD3 MicroBeads (Miltenyi Biotec, Auburn, CA). Cells were stained in the dark for 15 min with the following anti-human Abs: CD45RO PE-Cy7 (BD Biosciences, San Jose, CA), CD3 Alexa Fluor 700 (BD Biosciences), CD62L-PE (BD Biosciences), CD45RA-allophycocyanin (BD Biosciences), CD8-Pacific Blue (BD Biosciences), CD4 allophycocyanin-Cy7 (BD Biosciences), and LIVE/DEAD Aqua fluorescent reactive dye (Invitrogen, Grand Island, NY).
CD8⁺ T cell subsets were isolated using the BD FACSAria cell-sorting system (BD Biosciences), including CD8⁺CD45RA⁻CD45RO⁺ (for CD8⁺ memory), CD8⁺CD45RA⁺CD45RO⁻CD62L^hi (CD8⁺ naive), and CD8⁺CD45RA⁺CD62L^lo/− (CD8⁺ T_EMRA). FlowJo (TreeStar, Ashland, OR) analysis was used to determine the proportions of the different subsets as a fraction of total CD8⁺ T cells.

Plates were coated with SIVmac239 gp140 and incubated overnight at 4°C. All coated wells were washed and blocked with 5% BSA in PBS for 1 h at 37°C. Plates were then washed and diluted plasma samples were added into the corresponding wells for 1 h at 37°C. Simultaneously, CD107a PE Cy5 (BD Biosciences, #555802), brefeldin A (Sigma, #B7651), and GolgiStop (BD Biosciences, #554724) were incubated with human NK cell culture for 2 h. After both incubations were completed, we added the stimulated NK cells to the plates (50,000 cells per well), and incubated them for 5 h at 37°C. Next, NK cells were transferred to V-bottom plates containing CD56 PE Cy7 (BD Biosciences, # 557747), CD16 APC Cy7 (BD Biosciences, #55775), and CD3 Alexa Fluor 700 (BD Biosciences, #557943) and incubated for 15 min at room temperature. Plates were then washed and resuspended in PBS. Samples were acquired using a LSRII (BD Biosciences). Human NK cells were isolated using a RossetteSep NK cell enrichment kit (StemCell Technologies, #15065). NK cells were defined as CD3⁻/CD16⁺ and/or CD56⁺. Ab-dependent NK degranulation was calculated as the percentage of NK cells positive for CD107a.

CD3-Alexa Fluor 700, CD4-BV605, CD8-APCy7, CD38-APC, TIGIT-PE-Cy7, CD45RO-PECF594, CCR7-BV421, PD1-PE, ICOS-PcPCy5.5 and CXCR5-AF488 (BD Biosciences).
Cells were washed twice and run/sorted on a BD FACS Aria III. Samples from 12 study participants were selected based on the magnitude of their cTfh response from d0 to d7 to undergo bulk sorting, with samples from four of these participants also undergoing single cell sorting. Sorted cells had 100U/mL of RNaseOUT added prior to sorting. Bulk sorted cells were centrifuged with the supernatant removed prior to freezing at -80°C. Data were analysed with FlowJo 10.1 (TreeStar). Activated and resting cTfh cells (CD4⁺CXCR5⁺PD-1⁺) were defined as CD38⁺ICOS⁺ and CD38^-ICOS^-, respectively (19 (link), 22 (link)). Activated plasmablasts were defined as CD19⁺CD27⁺CD38⁺. An example of flow gating for activation and sorting is depicted in Supplementary Figure 8.